The overall objective of this proposal is to define the pathophysiology of the autosomal dominant transthyretin amyloidoses. These diseases which affect a number of kindreds worldwide and with large families in the United States are manifest as peripheral neuropathy, nephropathy, and cardiomyopathy. They are late onset diseases, but are associated with high degrees of morbidity and mortality. To date over 40 variant forms of transthyretin (TTR)each with a single amino acid substitution have been found to be associated with this autosomal dominant disease. How these mutations lead to amyloid fibril formation and disease expression is not. Considerable progress has been made in determining the structural changes of transthyretin which are related to the formation of amyloid. These studies include high resolution structures of the methionine 30 transthyretin, which is the most prevalent amyloid producing TTR variant, the serine 84 TTR and the alanine 60 TTR. In addition, the nonamyloid threonine 109 TTR structure has been determined for comparison. To date, however, no unifying structural factor has been found which explains amyloid fibril formation in all of these syndromes. Preliminary metabolic studies in the last funding period support the hypothesis that increased catabolism of the variant transthyretins may be related to generation of amyloid deposits. The Specific Aims are designed to test the hypothesis that the single amino acid substitutions associated with Amyloidosis cause: 1) Changes in metabolism of the variant molecule compared to normal; and 2) These changes are secondary to the structural perturbations in the variant proteins. A preliminary kinetic model of TTR metabolism has been formulated and this will be tested by the addition of more subjects into the metabolic studies. Additional variant forms of TTR will be expressed by recombinant DNA technology and studied by X-ray crystallography for analyzing the relationship of structure to the function of amyloid fibril formation. The X-ray structure of murine TTR, which does not form amyloid will be determined. A new Specific Aim will explore means of altering the expression of transthyretin in established cell culture systems. The experimental Design is to determine the effect of both protein concentration and antisense oligonucleotides on the expression of TTR by murine hepatocytes and human hepatoma cells in tissue culture. These experiments are directly aimed at devising means of altering expression of variant TTR proteins and, therefore, preventing development of the amyloid disease.